All reagents used in this study were of analytical grade. Anhydrous sodium sulfate (Na₂SO₄), methanol, dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, acetate buffer, Trolox, ascorbic acid, ellipticine, and dexamethasone were purchased from Sigma-Aldrich (USA). Sulforhodamine B (SRB), N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, phosphoric acid, and lipopolysaccharide (LPS) were obtained from REAGEN LLC (USA). Dulbeccoʼs modified Eagleʼs medium (DMEM) was sourced from Gibco (USA). The SK-LU-1 (human lung carcinoma), HepG2 (hepatocellular carcinoma), and RAW 264.7 macrophage cell lines were kindly provided by Prof. Domenico Delfino, University of Perugia, Italy. All other chemicals and solvents were procured from standard commercial suppliers.

Fresh leaves of C. thorelii were collected in February 2025 from Ninh Van, Ninh Hoa, Khanh Hoa, Vietnam (12°21'48.9"N, 109°16'07.8"E). The collection was conducted during the spring season in Central Vietnam, when C. thorelii exhibits robust vegetative growth and optimal essential oil accumulation. The botanical identification was performed by Dr. Anh Tuan Le of the Vietnam National Museum of Nature, Mien Trung Institute for Scientific Research. A voucher specimen, assigned the code CT-22025, was deposited at the herbarium of the University of Education, Hue University. The collected leaves were mature, characterized by their fully expanded, dark green appearance, indicating peak metabolic activity suitable for essential oil extraction. Based on local ecological observations, the flowering period of C. thorelii in this region typically occurs, suggesting that the February collection preceded the flowering phase to maximize leaf biomass and essential oil yield.

A Clevenger-type apparatus was used to distill 350 g of fresh C. thorelii leaves for four hours, following the protocol outlined in the Vietnamese Pharmacopoeia V (Ministry of Health, 2019). The entire 350 g of fresh material was processed in a single run to ensure consistency. Fresh leaves were chosen to preserve volatile terpenoid compounds, such as β-selinene and 1,8-cineole, which are prone to degradation during drying, as supported by studies on Croton species indicating higher essential oil quality from fresh material. The extracted essential oil was dried over anhydrous Na₂SO₄ at 5°C prior to further analysis. The yield was calculated based on the dry weight of the material to standardize comparisons with literature data, which typically report yields on a dry weight basis. To determine the dry weight, a separate 50 g sample of fresh leaves was oven-dried at 50°C until constant weight, revealing a moisture content of approximately 70%. Using this moisture content, the dry weight equivalent of the 350 g of fresh leaves was estimated to be 105 g.

GC-MS was employed to study the essential oilsʼ chemical constituents. The Shimadzu GCMS-QP2010 Plus system (Japan) had a fused silica Equity-5 capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, USA) [13, 14]. The chromatographic parameters were set as follows: injector and interface temperatures were maintained at 270°C, and helium was utilized as the carrier gas at a constant flow rate of 1.2 mL/min. From 50°C (held for 3 min) to 250°C (held for 12 min) and finally to 270°C (held for 20 min), the oven temperature was programmed to rise at a rate of 4°C/min. At an intake pressure of 90.0 kPa, 1.0 µL of each sample was injected with a split ratio of 30:1. With an electron ionization voltage of 70 eV, a detection voltage of 0.80 kV, and a scan rate of 0.5 scans per second, the mass spectrometer was able to measure masses between 40 and 500 amu. Retention indices (RIs), which were determined using a homologous sequence of n-alkanes (C₇–C₄₀), were compared with reference data from Adamsʼ library in order to identify the compounds [15]. The relative abundance of each component was estimated based on its peak area percentage. In order to guarantee reproducibility, every analysis was carried out in triplicate.

Mouse macrophage RAW 264.7 cells were used for anti-inflammatory evaluation, while human lung carcinoma (SK-LU-1) and hepatocellular carcinoma (HepG2) cell lines were employed for cytotoxicity assays. The SK-LU-1 and HepG2 cell lines were obtained from a certified cell bank and had been authenticated by short tandem repeat (STR) profiling by the supplier prior to distribution. All cells were cultured in DMEM supplemented with appropriate nutrients and 10% fetal bovine serum (FBS), and maintained at 37°C in a humidified atmosphere containing 5% CO₂. Cells were subcultured for at least two passages before being used in subsequent experiments.

The cytotoxic activity of essential oils extracted from the leaves of C. thorelii was evaluated using the SRB assay, following a spectrophotometric method [16]. Two cancer cell lines were tested: lung carcinoma (SK-LU-1) and hepatocellular carcinoma (HepG2), kindly provided by Prof. Domenico Delfino, University of Perugia, Italy. The cells were cultured in DMEM supplemented with L-glutamine, sodium pyruvate, NaHCO₃, 10% FBS, and 1% penicillin-streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) under conditions of 37°C and 5% CO₂. Subculturing was performed at a 1:3 ratio every 3–5 days, depending on cell growth. For the assay, cells were seeded in 96-well plates at a density of 5 × 10⁴ cells per well in 200 µL of culture medium and incubated for 24 h to allow attachment. The medium was then replaced with fresh medium containing essential oil samples at concentrations of 0.8, 4, 20, and 100 µg/mL. After 72 h of incubation at 37°C in a humidified 5% CO₂ atmosphere, the cells were fixed with 20% trichloroacetic acid (TCA) for 1 h at 4°C, washed with distilled water, and stained with 0.4% (w/v) SRB dye in 1% acetic acid for 30 min. Excess dye was removed by washing with 1% acetic acid, and bound dye was dissolved in 10 mM Tris base (pH 10.5). Optical density (OD) was measured at 540 nm using an ELISA plate reader. DMSO was used as the negative control at concentrations of 0.08, 0.4, 2, and 10 µg/mL, matching the sample concentration range. Ellipticine, used as the positive control, was tested at concentrations ranging from 0.1 to 10 µg/mL. The percentage of cell growth inhibition was calculated using the formula:

I n h i b i t i o n   %   = 100 - O D s a - O D d 0 O D n e g - O D d 0 × 100

Where OD_sa represents OD_{540 nm} of the samples (the oils or reference) treated wells after 72 h incubation, OD_neg refers to OD_{540 nm} of the DMSO negative control wells after 72 h incubation, OD_d0 refers to OD_{540 nm} of the control for the initial time point (Day 0). IC₅₀ values, representing the concentration required to inhibit 50% of cell growth, were determined using a nonlinear regression model (four-parameter logistic curve) in GraphPad Prism software (GraphPad Software, USA). All experiments were performed in triplicate to ensure reproducibility and accuracy.

To evaluate the essential oilsʼ antioxidant capacity, the DPPH (Sigma-Aldrich, USA) radical scavenging assay was employed [17]. A 0.25 mM DPPH solution was prepared in methanol, and 100 μL of this solution was combined with 1.4 μL of the essential oil sample (dissolved in DMSO) at concentrations ranging from 4 to 500 μg/mL. The mixtures were incubated in a 96-well microplate at 25°C for 30 min (in the dark). After incubation, the absorbance was recorded at 517 nm using a microplate reader (BioTek, USA). The following formula was used to determine the percentage of DPPH radical inhibition:

I n h i b i t i o n   %   = X - Y X × 100

Where X is the absorbance of the control (without sample) and Y is the absorbance in the presence of the test or reference sample. All measurements were conducted in triplicate. Ascorbic acid was used as the positive control, and IC₅₀ values were calculated using GraphPad Prism software.

The ABTS assay (Sigma-Aldrich, USA), which was modified slightly from the Saeed et al. [18] procedure, was used to assess the test substanceʼs antioxidant capacity. The following is how the methodology went: deionized water was used to dilute the sample until it reached concentrations of 10,000, 2,000, 400, and 80 µg/mL. The reference standard, Trolox, was produced similarly in deionized water at concentrations of 2,000, 400, 80, and 16 µg/mL. An ABTS solution (7 mM) was combined with potassium persulfate (2.45 mM) and allowed to stand at room temperature in the dark for 16 h. To get an OD of 0.70 ± 0.02 at 734 nm, the resultant ABTS^•+ solution was diluted with acetate buffer before analysis. Following that, 100 µL of each diluted sample was combined with 1,900 µL of the ABTS^•+ solution, resulting in final well concentrations of 500, 100, 20, and 4 µg/mL. A 1% DMSO solution served as the negative control, while wells containing solely deionized water functioned as blanks. A BioTek 96-well plate reader was used to capture absorbance measurements at 734 nm (BioTek, USA). The proportion of ABTS^•+ radical scavenging activity was determined using the formula below: A is the OD of the sample well minus the OD of the blank, and A₀ is the OD of the control well minus the OD of the blank.

I n h i b i t i o n   %   = A 0 - A A 0 × 100

The IC₅₀ value, which is the concentration required to block 50% of the ABTS^•+ radicals, was used to express the samplesʼ antioxidant capacity. All measurements were performed in triplicate (n = 3) to ensure statistical robustness, and results are reported as mean ± standard deviation (SD).

In this study, we combined the two methods (DPPH and ABTS assays) to provide a more comprehensive assessment and a more complete picture of the essential oilʼs antioxidant activity by accounting for different reaction mechanisms and sample solubilities.

RAW 264.7 macrophage cells were plated at a density of 2 × 10⁵ cells per well in 96-well plates [19]. The culture medium was replaced with DMEM, which is devoid of FBS, during a 24-hour incubation period. Following that, the cells were exposed to LPS (1.0 µg/mL) and essential oils at concentrations ranging from 5 to 100 µg/mL for 24 h in order to stimulate the generation of nitric oxide (NO). To evaluate NO levels, nitrite (NO₂⁻) buildup was quantified using the Griess reagent system (USA). 50 µL of 0.1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride and 50 µL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid were mixed with 100 µL of culture supernatant in a 96-well plate. After 10 min of incubation at 25°C, absorbance at 540 nm was measured using an RNE-9002 ELISA reader (REAGEN LLC, USA). A standard curve was used to calculate the nitrite levels.

I n h i b i t i o n   %   = 100 - N O c o n c e n t r a t i o n   i n   s a m p l e N O c o n c e n t r a t i o n   i n   n e g a t i v e   c o n t r o l × 100

Inhibition (%) was the formula used to determine the percentage of NO inhibition. The positive control used was dexamethasone (Sigma-Aldrich, USA). GraphPad Prism software was used to determine the IC₅₀ values for each experiment, which were carried out in triplicate.

Experimental data are presented as the mean ± SD, calculated from the number of replicates. IC₅₀ values were calculated using nonlinear regression analysis based on dose-response curves. These curves were generated by plotting the percentage of inhibition versus the logarithm of sample concentrations, with the IC₅₀ defined as the concentration that produced 50% inhibition.

Hydrodistillation of fresh C. thorelii leaves produced a dark yellow essential oil with a yield of 0.21% (v/w). GC-MS analysis identified 59 compounds, representing 99.5% of the total composition (see Table 1 and Figure S1). The oil was mainly composed of sesquiterpene hydrocarbons (44.5%), oxygenated monoterpenes (41.1%), and oxygenated sesquiterpenes (9.4%). Minor components included monoterpene hydrocarbons (3.9%), oxygenated diterpenes (0.4%), and non-terpenic compounds (0.2%). The major constituents were β-selinene (22.0%), 1,8-cineole (20.7%), linalool (11.2%), and (E)-caryophyllene (9.5%). Other significant compounds (≥ 1.0%) included α-terpineol, bicyclogermacrene, α-humulene, borneol, caryophyllene oxide, spathulenol, β-bourbonene, δ-cadinene, and terpinen-4-ol.

This study investigated the cytotoxic effects of C. thorelii leaf essential oil on SK-LU-1 and HepG2 cell lines using the SRB assay. The results demonstrated notable inhibitory activity, with IC₅₀ values of 54.52 ± 1.40 μg/mL for SK-LU-1 and 48.29  ±  2.09 μg/mL for HepG2.

The antioxidant activity of C. thorelii essential oil was assessed using DPPH and ABTS assays. It showed no significant activity in the DPPH assay (IC₅₀ > 500 µg/mL) and weak activity in the ABTS assay (IC₅₀ = 453.85 ± 15.87 µg/mL).

In LPS-stimulated RAW 264.7 macrophages, the essential oil from C. thorelii leaves exhibited moderate anti-inflammatory activity in the nitric oxide (NO) production assay. At 100 µg/mL, the essential oil suppressed NO generation by 61.46 ± 2.37%; however, this effect was associated with reduced cell viability (57.91 ± 2.98%), indicating possible cytotoxicity at higher concentrations. In contrast, the positive control, dexamethasone, demonstrated stronger inhibition (86.59 ± 1.12%) while maintaining higher cell viability (92.71 ± 2.45%). The inhibitory effect of C. thorelii essential oil decreased in a dose-dependent manner, with NO inhibition rates of 10.57 ± 1.01%, 5.85 ± 0.61%, and 1.81 ± 0.12% at 50, 25, and 12.5 µg/mL, respectively. At 50 µg/mL, cell viability improved to 83.47 ± 2.20%, suggesting reduced cytotoxic effects at lower concentrations. The IC₅₀ value of the essential oil could not be determined within the tested concentration range, as 50% inhibition was not achieved under non-cytotoxic conditions. Conversely, dexamethasone exhibited an IC₅₀ value of 13.24 ± 1.23 µg/mL. Although the anti-inflammatory potency of C. thorelii essential oil was lower than that of the standard drug, its activity may be attributed to the synergistic effects of its bioactive constituents.

A complex phytochemical profile that distinguishes this essential oil from those of other Croton species reported in the literature and reflects both species-specific variability and regional impacts within Vietnam was found in the essential oil isolated from the fresh leaves of C. thorelii in this investigation. Comparatively, the essential oil of C. tonkinensis leaves from Vietnam, as reported by [20], contained significant amounts of (E)-caryophyllene (10.1%), linalool (7.8%), and α-humulene (7.1%), alongside bicycloelemene (8.0%) and β-bisabolene (9.6%). While (E)-caryophyllene and linalool are common to both species, C. thorelii exhibits a higher prevalence of oxygenated monoterpenes, such as 1,8-cineole (20.7%), which is absent or minimal in C. tonkinensis. Similarly, C. cascarilloides from the same study was dominated by hydrocarbon compounds like (E)-caryophyllene (13.5%) and α-humulene (5.9%), with β-selinene (6.7%) present but at a much lower concentration than in C. thorelii (22.0%) [20]. In contrast, C. chevalieri showed a distinct profile with non-terpenoid compounds like cyclohexanone (6.8%) and benzyl benzoate (18.8%), which are negligible in C. thorelii (0.2% non-terpenoids) [20].

The prominence of (E)-caryophyllene in Croton species is a recurring theme across studies. For instance, C. delpyi leaves from Vietnam yielded (E)-caryophyllene (54.34%) and α-humulene (18.19%) as the major components [21], far exceeding the 9.5% and 2.7% observed in C. thorelii, respectively. This suggests a stronger sesquiterpene hydrocarbon focus in C. delpyi compared to the balanced sesquiterpene-monoterpene profile of C. thorelii. Likewise, C. hirtus aerial parts from Vietnam contained α-humulene (8.5%), germacrene D (11.6%), and (E)-caryophyllene (32.8%) [10], aligning with C. thorelii in terms of sesquiterpene presence but lacking the high oxygenated monoterpene content seen in our study.

Its constituents, such as linalool (15.05% vs. 11.2%), (E)-caryophyllene (7.91% vs. 9.5%), bicyclogermacrene (7.36% vs. 3.0%), and 1,8-cineole (6.53% vs. 20.7%), are rather similar to those of C. thorelii [22]. However, the higher 1,8-cineole content in C. thorelii underscores a greater oxygenated monoterpene contribution, possibly linked to differences in plant parts (leaves vs. stems) or environmental factors. In contrast, C. zehntneri leaves from Brazil exhibited a markedly different composition, with estragole dominating at 84.7% or 76.8%, alongside minor amounts of 1,8-cineole (7.0%) and spathulenol (5.6%) [23, 24]. This phenylpropanoid-heavy profile contrasts sharply with the terpene-rich essential oil of C. thorelii.

Further afield, C. matourensis leaves from Brazil contained (E)-caryophyllene (12.41%) and thunbergol (11.74%) [25], while C. piauhiensis leaves featured (E)-caryophyllene (21.58%) and D-limonene (13.47%) [26]. Neither species showed the high levels of β-selinene or 1,8-cineole found in C. thorelii. Similarly, C. hirtus leaves from Brazil varied by location, with spathulenol (26.7%) or (E)-caryophyllene (27.9–37.3%) as major constituents [27], compared to 1.7% and 9.5% in C. thorelii, respectively. Both C. monteverdensis and C. niveus bark essential oils from Costa Rica were high in α-pinene (17.1% and 14.4%) and 1,8-cineole (11.6% in C. niveus) [28], which is consistent with the occurrence of 1,8-cineole in C. thorelii but differs in terms of sesquiterpene dominance.

The chemical diversity among Croton species is further exemplified by several notable cases. For example, linalool accounted for 34.9% of C. micradenus leaf essential oil from Cuba [29], which is significantly more than the 11.2% observed in C. thorelii. In contrast, methyl eugenol, which was completely missing from our analysis, was abundant in C. malambo leaves from Venezuela (94.2%) [30]. Similarly, C. huberi, also from Venezuela, contained substantial amounts of germacrene D (16.1%) and (E)-caryophyllene (18.3%) [31]. Meanwhile, genotypes of C. tetradenius from Brazil exhibited either camphor (13.95%) or p-cymene (17.55%) as dominant components [32]. None of these major constituents were significant in C. thorelii, where their presence did not exceed 1.0%.

The elevated β-selinene (22.0%) in C. thorelii is particularly notable, as it exceeds levels reported in other Croton species, such as 6.7% in C. cascarilloides [20]. This, combined with substantial 1,8-cineole and linalool, suggests a unique chemotype influenced by genetic factors, soil conditions, or climate in Central Vietnam. Variations across Croton species may also reflect differences in harvest time, plant part, or extraction methods (e.g., hydro-distillation vs. steam distillation). These findings underscore the chemical diversity within the genus Croton and highlight C. thorelii as a distinct source of terpene-rich essential oil with potential pharmacological applications, warranting further investigation into its biosynthetic pathways and ecological roles.

SK-LU-1 and HepG2 cell lines were both significantly cytotoxically affected by the essential oil extracted from C. thorelii leaves, with IC₅₀ values of 54.52 ± 1.40 µg/mL and 48.29 ± 2.09 µg/mL, respectively (Table 2). The essential oil showed substantial cytotoxic potential, inhibiting cell proliferation in both cell lines by over 90% at the highest tested concentration of 100 µg/mL. However, at lower concentrations (20, 4, and 0.8 µg/mL), the inhibition decreased significantly, ranging from 7% to 25%, indicating a dose-dependent response. The chemical composition of C. thorelii essential oil, dominated by sesquiterpene hydrocarbons, oxygenated monoterpenes, and oxygenated sesquiterpenes, with major constituents such as β-selinene (22.0%), 1,8-cineole (20.7%), linalool (11.2%), and (E)-caryophyllene (9.5%), likely contributes to its bioactivity. These findings align with the broader literature on Croton species, though the potency and specificity of cytotoxic effects vary across species and cell lines.

Comparatively, the essential oil from C. delpyi leaves, also sourced from Vietnam, displayed significantly higher cytotoxicity against HepG2 cells (IC₅₀ = 2.09 ± 0.11 µg/mL) and HeLa cells (IC₅₀ = 3.51 ± 0.15 µg/mL) [21]. The C. delpyi essential oil was rich in (E)-caryophyllene (54.34%) and α-humulene (18.19%), with smaller amounts of linalool (3.22%), contrasting with the more balanced sesquiterpene-monoterpene profile of C. thorelii. The increased (E)-caryophyllene concentration of C. delpyi essential oil, a substance known for its cytotoxic qualities through mechanisms like apoptosis induction and cell cycle arrest, may be the cause of its greater potency. Although C. thorelii contains (E)-caryophyllene (9.5%), its lower concentration, combined with the presence of 1,8-cineole and β-selinene, may result in a more moderate cytotoxic effect.

In contrast, estragole, the primary component (84.7%) of C. zehntneri essential oil from Brazil, showed no significant inhibition against MCF-7, HEP-2, and NCI-H292 cell lines, despite exhibiting toxicity against Artemia salina (LC₅₀ = 4.54 µg/mL) [23]. The lack of cytotoxicity of C. zehntneri essential oil in human cell lines, despite its high estragole content, suggests that phenylpropanoids like estragole may not be as effective against cancer cells as terpenoids, which dominate C. thorelii essential oil. This highlights the role of terpenoid constituents, such as linalool and (E)-caryophyllene, in C. thorelii, in driving its cytotoxic activity.

The essential oil from C. matourensis leaves exhibited IC₅₀ values of 28.5 µg/mL (HepG2), 23.3 µg/mL (MCF-7), 28.9 µg/mL (HCT116), 17.8 µg/mL (HL-60), and 25.8 µg/mL (MRC-5) against various cell lines [25]. The C. matourensis essential oil contained thunbergol (11.74%) and (E)-caryophyllene (12.41%), with a higher (E)-caryophyllene content than C. thorelii but lacking significant oxygenated monoterpenes like 1,8-cineole. C. matourensis has a lower IC₅₀ value (28.5 µg/mL) against HepG2 than C. thorelii (48.29 µg/mL), indicating increased efficacy, maybe as a result of the synergistic actions of its sesquiterpene and diterpenoid (thunbergol) components. However, C. thorelii’s broader terpenoid profile may contribute to its consistent activity across both SK-LU-1 and HepG2 cell lines.

Likewise, the leaf essential oil of C. malambo from Venezuela, which is rich in methyl eugenol (94.2%), demonstrated cytotoxic effects against MCF-7 cells (IC₅₀ = 72.84 µg/mL), while displaying lower activity against PC-3 and LoVo cell lines [30]. The IC₅₀ value for MCF-7 is higher than the IC₅₀ values of C. thorelii against SK-LU-1 and HepG2, indicating that C. thorelii essential oil is more potent against the tested cell lines. The high methyl eugenol content in C. malambo essential oil, a phenylpropanoid, contrasts with the terpenoid-rich composition of C. thorelii, further supporting the hypothesis that terpenoids like linalool, 1,8-cineole, and (E)-caryophyllene may be more effective cytotoxic agents in this context.

The cytotoxic activity of C. thorelii essential oil, while moderate compared to C. delpyi and C. matourensis, demonstrates its potential as an anticancer agent, particularly given its efficacy at higher concentrations (> 90% inhibition at 100 µg/mL). The presence of linalool and 1,8-cineole, known for their antiproliferative and apoptotic effects, likely contributes to this activity, alongside (E)-caryophyllene’s established role in cancer cell inhibition. The variability in potency across Croton species underscores the influence of chemical composition, which is shaped by genetic, environmental, and geographical factors. To help create new therapeutic treatments, more research is required to determine the precise mechanisms of action of C. thorelii essential oil and evaluate its possible effectiveness against a wider variety of cancer cell lines.

With an IC₅₀ value of more than 500 µg/mL in the DPPH assay (Table 3) and 453.85 ± 15.87 µg/mL in the ABTS assay (Table 4), the essential oil isolated from C. thorelii leaves had little antioxidant activity. The IC₅₀ values of 7.31 ± 0.41 µg/mL (DPPH) and 7.76 ± 0.42 µg/mL (ABTS) for the standard L-ascorbic acid were significantly lower than these values, suggesting that C. thorelii essential oil has a weaker capacity to scavenge radicals than the reference chemical. The mild antioxidant activity of C. thorelii essential oil is probably influenced by its chemical composition. The results could be explained by the lack of important phenolic substances, which are recognized for their strong antioxidant qualities.

In contrast, the essential oil extracted from the leaves of C. piauhiensis exhibited a wide range of antioxidant activity in several tests, with IC₅₀ values varying between 171.21 and 4,623.83 µg/mL [26]. Specifically, in the DPPH assay, C. piauhiensis essential oil had an IC₅₀ of 4,623.83 µg/mL (compared to quercetin, IC₅₀ = 2.71 µg/mL), which is significantly higher than C. thorelii’s IC₅₀ (> 500 µg/mL), indicating poorer DPPH radical scavenging capacity. However, C. piauhiensis obtained an IC₅₀ of 171.21 µg/mL in other tests, such as the β-carotene bleaching test (BCB), indicating greater activity in specific situations. The essential oil of C. piauhiensis consist of Germacrene D (9.56%), D-limonene (13.47%), and (E)-caryophyllene (21.58%). Since monoterpenes with allylic hydrogens are known to have protective effects against lipid peroxidation, the presence of monoterpene hydrocarbons like D-limonene, which is present in trace amounts in C. thorelii (0.6%), may be a factor in its improved performance in the BCB assay.

In the DPPH assay, the essential oils of C. argyrophylloides, C. nepetifolius, and C. jacobinensis exhibited weaker antioxidant activity, showing IC₅₀ values of 12.55, 22.11, and 24.96 µg/mL, respectively, which were higher than those of the standards thymol and butylated hydroxytoluene, with IC₅₀ values of 3.47 µg/mL and 5.16 µg/mL [33]. Although the chemical compositions of these essential oils were not specified, prior work by the same group highlighted that phenolic compounds, monoterpene hydrocarbons (e.g., terpinolene, α-terpinene, γ-terpinene), and allylic alcohols contribute significantly to antioxidant activity [9]. In C. thorelii, the low monoterpene hydrocarbon content (3.9%) and absence of phenolics likely underlie its weaker DPPH performance compared to these species.

With an IC₅₀ of less than 63.59 µg/mL, the essential oil from C. cajucara, which has a high concentration of 7-hydroxycalamenene (28.4%–37.5%), demonstrated greater antioxidant activity in the DPPH assay [34]. The presence of 7-hydroxycalamenene, a sesquiterpene with a phenolic-like structure, likely accounts for its superior radical scavenging capacity. In contrast, C. thorelii lacks phenolic or phenolic-like compounds, relying instead on terpenoids like linalool (11.2%) and 1,8-cineole (20.7%), which offer only moderate antioxidant effects due to their ability to donate hydrogen atoms.

The IC₅₀ of C. ferrugineus essential oil in the ABTS experiment was 901 ± 20 µg/mL [35], which is higher than that of C. thorelii (453.85 ± 15.87 µg/mL), indicating that C. thorelii has a relatively better ABTS radical scavenging capacity. The chemical composition of C. ferrugineus was not detailed, but its lower antioxidant activity suggests a possible lack of effective radical scavengers like linalool, which is present in C. thorelii and known to contribute to ABTS scavenging through its hydroxyl group.

Finally, the essential oil from the stem bark of C. urucurana exhibited an IC₅₀ of 3.21 mg/mL (3,210 µg/mL) in the DPPH assay [36], a value much higher than C. thorelii’s IC₅₀ (> 500 µg/mL), indicating significantly weaker antioxidant activity. The composition of C. urucurana essential oil was not specified, but its high IC₅₀ suggests a limited presence of active antioxidant compounds, contrasting with C. thorelii’s more diverse terpenoid profile, which includes linalool and 1,8-cineole, both known for moderate radical scavenging properties.

C. thorelii essential oilʼs low monoterpene hydrocarbon concentration and absence of phenolic components, which are both necessary for potent radical scavenging, are the reasons for its weak antioxidant activity, especially in the DPPH experiment. However, its moderate ABTS activity may be driven by oxygenated monoterpenes like linalool and 1,8-cineole, which can stabilize radicals through hydrogen donation. The variability in antioxidant performance across Croton species highlights the influence of chemical composition, which is shaped by genetic, environmental, and geographical factors. While C. thorelii essential oil does not exhibit the potency of phenolic-rich essential oils like C. cajucara, its moderate ABTS activity suggests potential for use in combination with other antioxidants, such as in natural preservative systems, warranting further exploration of its synergistic effects.

In LPS-stimulated RAW 264.7 macrophage cells, the essential oil from C. thorelii leaves showed considerable anti-inflammatory efficacy in the nitric oxide (NO) generation assay (Table 5). At a concentration of 100 µg/mL, the essential oil inhibited NO production by 61.46 ± 2.37%, though this was accompanied by a reduced cell viability of 57.91 ± 2.98%, suggesting potential cytotoxicity at higher concentrations. In comparison, the positive control, dexamethasone, achieved 86.59 ± 1.12% inhibition with a higher cell viability of 92.71 ± 2.45%. NO inhibition was dose-dependently reduced at lower doses by C. thorelii essential oil, with values of 10.57 ± 1.01% at 50 µg/mL, 5.85 ± 0.61% at 25 µg/mL, and 1.81 ± 0.12% at 12.5 µg/mL, while cell viability improved to 83.47 ± 2.20% at 50 µg/mL (viability data for 25 and 12.5 µg/mL were not available). The IC₅₀ for C. thorelii essential oil could not be determined, whereas dexamethasone exhibited an IC₅₀ of 13.24 ± 1.23 µg/mL. Although C. thorelii essential oilʼs potency is limited when compared to the standard, its chemical composition probably plays a role in its anti-inflammatory actions.

In LPS-stimulated RAW 264.7 macrophages, essential oils obtained from C. kongensis stems collected in Nhu Xuan and Thuong Xuan districts (Thanh Hoa province, Vietnam) inhibited NO production, yielding IC₅₀ values of 105.71 ± 0.96 µg/mL and 94.93 ± 1.31 µg/mL, respectively [22]. The C. kongensis essential oil contained 1,8-cineole (6.53%), (E)-caryophyllene (7.91%), bornyl acetate (9.52%), and linalool (15.05%), sharing some compositional similarities with C. thorelii, particularly in linalool and 1,8-cineole content. Although an IC₅₀ for C. thorelii was not determined, its inhibition of > 61% at 100 µg/mL suggests comparable or slightly better activity at this concentration than C. kongensis, which required a similar concentration range to achieve its IC₅₀. The presence of 1,8-cineole and linalool in both essential oils likely contributes to their anti-inflammatory effects, as these compounds are known to suppress pro-inflammatory pathways, including NO production.

The essential oil from C. rhamnifolioides leaves, with 1,8-cineole as its major constituent (41.33%), exhibited significant anti-inflammatory activity in vivo [37]. The essential oil decreased Croton essential oil-induced edema by 42.1% at 20 mg/mL, but 1,8-cineole by itself (8.26 mg/mL) reduced it by 34.9%. Furthermore, the essential oil and 1,8-cineole (10.33–82.66 mg/kg) significantly decreased vascular permeability and paw edema brought on by carrageenan, dextran, histamine, and arachidonic acid at doses of 25–200 mg/kg. The high 1,8-cineole content in C. rhamnifolioides essential oil, compared to 20.7% in C. thorelii, likely enhances its anti-inflammatory efficacy, as 1,8-cineole is known to inhibit pro-inflammatory cytokines and mediators like NO. The in vitro NO inhibition by C. thorelii essential oil (> 61% at 100 µg/mL) aligns with the anti-inflammatory potential of 1,8-cineole, though its lower concentration and the presence of other terpenoids like β-selinene may dilute its overall potency compared to C. rhamnifolioides.

A study investigating the essential oil of C. rhamnifolioides complexed with β-cyclodextrin (COEFC) revealed anti-inflammatory effects in vivo, underscoring the critical role of 1,8-cineole [37]. In line with the results of the uncomplexed essential oil, all tested doses of COEFC decreased vascular permeability and acute paw edema brought on by carrageenan and dextran [37]. The complexation probably increased 1,8-cineoleʼs bioavailability, which strengthened its anti-inflammatory properties. In contrast, C. thorelii essential oil, tested in vitro, showed a dose-dependent NO inhibition but with reduced cell viability at higher concentrations, suggesting that its anti-inflammatory potential may be limited by cytotoxicity, a factor not reported in the C. rhamnifolioides studies.

The moderate anti-inflammatory properties of C. thorelii leaf essential oil are due to the presence of 1,8-cineole and linalool, which are known to influence inflammatory pathways by lowering the production of NO and the expression of cytokines. The presence of (E)-caryophyllene (9.5%), a sesquiterpene with documented anti-inflammatory properties via cannabinoid receptor pathways, may also contribute to its activity. However, the high β-selinene content (22.0%), which lacks significant anti-inflammatory activity, may dilute the overall effect compared to essential oils with higher concentrations of active compounds like 1,8-cineole in C. rhamnifolioides. The variability in anti-inflammatory potency across Croton species highlights the influence of chemical composition, which is shaped by genetic, environmental, and geographical factors. The dose-dependent response of C. thorelii essential oil, coupled with its cytotoxicity at higher concentrations, suggests that further optimization, such as complexation or combination with other agents, could enhance its therapeutic potential. The observed > 61% NO inhibition at 100 µg/mL, while promising, coincides with a cell viability of only 57.91%, which may indicate nonspecific toxicity rather than a specific anti-inflammatory effect. This suggests that the anti-inflammatory activity at this concentration could be confounded by cytotoxic effects, particularly given the lower viability compared to dexamethasone (92.71%). At lower concentrations (e.g., 50 µg/mL), where viability improves to 83.47%, the inhibition drops significantly (10.57%), further supporting the possibility of toxicity-driven effects at higher doses. This limitation highlights the need for further investigation, such as in vivo studies or testing at a broader range of concentrations, to differentiate between specific anti-inflammatory activity and nonspecific cytotoxicity, and to optimize the therapeutic window of C. thorelii essential oil.

The essential oil from C. thorelii leaves, collected in Central Vietnam, exhibits a terpene-rich composition with potential cytotoxic and anti-inflammatory properties. While its antioxidant activity is limited, the essential oil shows promise for therapeutic applications, particularly in anticancer and anti-inflammatory contexts. Further optimization and in vivo studies are recommended to explore its full potential.